Effect of Proteolytic Enzymes on Sedimentation Properties of Ribonucleoprotein Particles from Heart Muscle.

There is much support for the view that the functional unit of protein synthesis is an aggregate of ribosomes held together by a strand of messenger RNA.1-6 The concept derives from the observation that radioactive amino acids are preferentially incorporated into ribosomal aggregates (termed polyribosomes, polysomes, or ergosomes) and that treatment of the ribosomal aggregates with small amounts of ribonuclease leads to the formation of single ribosomes.2-5 In a study7-9 of the characteristics of ribonucleoprotein particles from rat heart muscle it was found that the particles when analyzed on linear sucrose gradients gave no distinct 70-80S peak, nor were there more than small amounts of intermediatesized aggregates present; instead, almost all the material sedimented in a pellet of S value greater than 400. The sedimentation pattern was not affected by ribonuclease treatment of the ribonucleoprotein particles prior to analysis, but chymotrypsin did increase the amount of 70-80S material. The latter observation suggested the possibility that in muscle the functional unit for protein synthesis might be a very large assemblage of ribonucleoprotein particles held together by protein, perhaps the nascent peptide being synthesized on the ribosomes, as well as by messenger RNA. Indeed, it seemed possible that in muscle more than one ribosome might be contributing to the synthesis of a single protein, an interpretation that accords with the suggestion that for the synthesis of structural protein of high molecular weight, or of enzymes composed of several subunits, polymerization of the polypeptide units occurs on the ribosomal assemblage. 10 It is the purpose of this paper to describe the results of controlled proteolytic digestion of ribonucleoprotein particles isolated from heart muscle on their sedimentation behavior in linear sucrose gradients. Treatment of ribonucleoprotein particles, labeled with radioactive leucine, with small amounts of chymotrypsin or trypsin has been found to lead to the disruption of the rapidly sedimenting material into smaller particles of 70-80S and, pari passu, by the release of radioactive material derived from the nascent peptide. We believe the finding to have significance for the mechanism of the synthesis of the protein structural units of the myofibril. Experimental Procedures.-Ribonucleoprotein particles were labeled by incubation of minced heart muscle from 14-18-day chick embryos in 5 vol of Eagle's minimal essential culture medium (spinner),I1 from which the C"2-leucine had been omitted; the medium contained 40 juc/ml of H3leucine (2.5 c/mmole). Incubation was for 15 min at 37°. After incubation the ribonucleoprotein particles were prepared by either of two procedures: treatment of the 105,000 X g sediment from a whole tissue homogenate with deoxycholate; or from the microsomal fraction. Both methods have been described in detail.9 The ribonucleoprotein particles were suspended in buffer (0.05 M Tris-HCl, pH 7.4; 0.005 M MgCl2; 0.1 M KCI) and an aliquot incubated for 30 min at O with or without the concentration of enzyme indicated in the figures. Liver ribosomes were prepared from microsomes in the customary manner.'2 The ribonucleoprotein particles were